ViralRecall

ViralRecall is a flexible command-line tool for predicting prophage and other virus-like regions in genomic data. This code is under development and likely to change as bugs are identified.

Dependencies

ViralRecall is written in Python 3.5.6 and requires biopython, matplotlib, numpy, and pandas. ViralRecall uses Prodigal and HMMER3 for protein prediction and HMM searches, respectively. Please ensure these tools are installed in your PATH before using. One a Unix system you should be able to install these tools with:

sudo apt install prodigal

and

sudo apt install hmmer

or if you don't have sudo priveleges, you can try with conda:

conda install prodigal -c bioconda

and

conda install hmmer3 -c bioconda

Installation

Please ensure you are using > Python 3.5.2 and have the appropriate python modules installed. If this is an issue please create a Python environment using conda (see here https://conda.io/projects/conda/en/latest/user-guide/tasks/manage-environments.html) ViralRecall was tested on Ubuntu 16.04 and should work on most Unix-based systems. To see the help menu use:

python viralrecall.py -h

Databases

Viralrecall uses two main HMM databases to analyze viral signatures in genomic data. The first is Pfam, which is a broad-specificity database that detects many protein families that are common in the genomes of cellular organisms. The Pfam database here has been modified to remove common viral protein families. The second HMM database is VOGDB, which is a comprehensive database of known viral protein families. The full VOG HMM database is quite large, and in some situations users may wish to use smaller sets to speed up runtime. We have created three VOG database that vary in size (all, large, and small). Users wishing to perform an exhaustive search should use the "all" database, while those preferring a faster search may wish to use the "small" database (the "large" database is an intermediate size option).

The database files are available for download from the Virginia Tech library system. To download and unpack, navigate to the folder that contains the viralrecall.py script and type:

wget -O hmm.tar.gz https://data.lib.vt.edu/downloads/8k71nh28c

and then

tar -xvzf hmm.tar.gz

Basic Usage

To test if ViralRecall will run properly type:

python viralrecall.py -i examples/test_seq.fna -p test_outdir -t 2 -db small -f

Results should be located in the test_outdir folder. The output folder will contain:

*.faa: The proteins predicted from the input file using Prodigal

*.full_annot.tsv: A full annotation table of the predicted ORFs.

*.prophage.annot.tsv: An annotation of only the viral regions (only present if some viral regions found)

*summary.tsv Summary statistics for the predicted viral regions.

*.pfamout Raw output of the Pfam HMMER3 search

*.vogout Raw output of the VOGDB HMMER3 search

Additionally, for each viral region viralrecall will print out .faa and .fna files for the proteins and nucleotide sequences for the regions found. Please be sure to use only .fna files as input.

Options

There are several parameters you can change in viralrecall depending on your preferences and the data you're analyzing. The important parameters that will influence the results are:

-s, --minscore This is the mean score that a genomic regions needs to have in order to pass the filter and get reported as a viral region. The score is calculated from the HMMER3 scores, with higher scores indicating more and better matches to the VOG database, and lower scores indicating more and higher matches to the Pfam database. The default is 10.

-w, --window Size of the sliding window to use for calculating moving averages. A smaller window may help predict short viral regions, but may split large viral regions into several pieces.

-m, --minsize Minimum size, in kilobases, of the viral regions to report.

-v, --minvog Minimum number of hits against the VOG database that must be recorded in a region in order for it to be reported (larger values == higher confidence).

For example, if we wanted to recover only long, well-defined viral regions we could use the following command:

python viralrecall.py -i examples/test_seq.fna -p testout -s 15 -m 30 -v 10

Here we are asking for only regions that have a mean score >= 15, are at least 30 kilobases long, and have at least 10 VOG hits.

If we want to quickly re-do the above analysis with different parameters, but without re-doing gene predictions and HMMER3 searches, we can use the -r flag:

python viralrecall.py -i examples/test_seq.fna -p testout -s 15 -m 30 -v 10 -r

This should re-calculate the results quickly and allow you to identify the most appropriate ones for your analysis.

Batch mode

If you have many sequences you wish to test you can put them all in a folder and use batch mode. Here the input (-i) should point to a folder with .fna files in it. Basic usage is:

python viralrecall.py -i examples/testfolder -p folderout -b

All of the output files should have their own folder in the folderout directory. You can also use the -b flag with the -r flag for quick re-calculations.

Citation

Citation for this tool is pending. If you plan on using this tool in an article please email Frank Aylward to ask the best way to cite this work at faylward at vt dot edu

Also, since this tool requires Prodigal and HMMER3, please cite thest tools as well. Their citations are:

Hyatt et al. “Prodigal: prokaryotic gene recognition and translation initiation site identification”. BMC bioinformatics, 2010. Eddy, "A new generation of homology search tools based on probabilistic inference". Genome Informatics, 2009.